The Role Of Mechanical Sensitive MicroRNA-199a-5p In The Degeneration Of Endplate Chondrocytes

Objective: To investigate the mechanism of mechanosensitive Micro RNA-199a-5p in the degeneration of endplate chondrocytes by establishing a strain-induced degeneration model of endplate chondrocytes in vitro.Methods: The endplate cartilage of cervical spondylosis was collected in patients with cervical spine fractures.The degeneration model of endplate chondrocytes was established by FX-5000 cell strain loading system using Intermittent cyclic mechanical tension(ICMT)at a conditons of 10% elongation?0.5 Hz and for 12 h per day.Micro RNAs were used to detect the expression of differentially expressed Micro RNAs after applied mechanical stimulation.Realtime RT-PCR was applied to verify the results of the microarrays.Target genes were predicted via Targetscan,Miranda,mi Rbase and mirdb biology software.Select the target Micro RNA199a-5p for experiments in vitro.Cell staining and the phenotypes of chondrocytes were observed after applied mechanical stimulation.The proliferation,apoptosis and cytoskeleton changes of endplate chondrocytes were detected by Alamar Blue?flow cytometry and phalloidin staining respectively.Dual luciferase Reporter gene method was used to verify the direct effect of Micro RNA-199a-5p on IKBKB target gene.And then Real-time RT-PCR and western blott methods were used to study the related genes type 2 collagen(COL2A1)?proteoglycan(ACAN)and SOX9 Gene and protein level of chondrocytes after treated with si RNA-IKBKB,mimic and inhibitor Micro RNA-199a-5p.Results: The expression of cartilage-associated markers COL2A1,ACAN and SOX9 in endplate chondrocytes decreased with the extension of stretching time,the synthesis level of endplate chondrocytes was significantly inhibited.ICMT can promote the proliferation of endplate chondrocytes and it also can change the morphology of chondrocyte cytoskeletal,but the apoptosis level of chondrocytes was not change significantly.Microarray chip screening of endplate chondrocytes showed that 9 micro RNAs were up-regulated and 16 micro RNAs were down-regulated after applied mechanical stimulation.IKBKB as a potential target gene of Micro RNA-199a-5p through informatics software analysis.Overexpression Micro RNA-199a-5p can significantly inhibited the expression of IKBKB.Overexpression(mimic)micro RNA-199a-5p and si RNA interference with IKBKB,the related genes level of chondrocytes(COL2A1,ACAN and SOX9)were increased;The degeneration of endplate chondrocyte can be significantly alleviated.Conclusion: ICM stimulation can include degeneration of endplate chondrocytes.The degeneration of endplate chondrocytes can be alleviated by both overexpression of Micro RNA-199a-5p and inhibition of its target gene IKBKB.